This proposal represents a continuation and expansion of the PI's initial objective of attempting to understand the molecular mechanisms responsible for hepatic fibrogenesis. One difficulty in their search for answers has been the lack of an adequate rodent model for the development of hepatic injury and fibrosis caused by ethanol. Thus, they propose to develop improved in vitro and in vivo methods of ethanol-induced ad liver injury and to employ the models to investigate the significance of two interrelated factors, tissue transglutaminase (tTG) and nuclear factor-kappa B (NF-kappaB), in the process of hepatic injury and fibrosis. Specific Aims: 1) to develop new models of ethanol-induced hepatic injury and fibrogenesis; 2) to delineate the role of NF-kappaB as a mediator of hepatic injury and fibrogenesis; 3) to assess how tTG affects hepatic injury and fibrogenesis; and, 4) to determine the relationship between the GTP-binding and cross-linking activities of tTG. Methods: Isolated calls from Osteogenic Disorder Shionogi (ODS) rats will be treated with ethanol to characterize aspects of the injury and activation process. The mechanisms by which ethanol injures the hepatocytes will be evaluated by malondialdehyde levels, Northern blot hybridization analysis of cytokines and tTG, and gel retardation assays of NF-kappaB. ODS rats will be fed the Lieber- DeCarli diet in an attempt to develop an improved model for chronic alcoholic liver disease. Once the models are established, attempts will be made to inhibit injury in the model systems with novel therapeutic agents. Attempt to determine pathophysiologic significance of NF-kappaB in the injury process will be under undertaken using a proteasome inhibitor which blocks the degradation of IkappaB. Mutagenesis directed to the NF-kappaB motif as well as analysis of other regulatory regions of the tTG promoter will be used to determine the expression a tTG in in vivo injury models. Specific a1-adrenergic receptor agonist and antagonist treatment of hepatocytes will be undertaken to investigate the interaction between the GTPase and cross-linking activity of tTG. Health Relatedness: It is hoped that by better understanding the molecular bases of alcoholic liver diseases, effective and rational therapy can be developed.